Interpretive Summary: Ustilago maydis, the causal agent of common smut of corn, is dimorphic, having a single cellular yeast stage and a multicellular filamentous phase. These stages are expressed at different pH. A specific molecular approach (suppression subtractive hybridization) was used to identify genes involved in pH induced differentiation between yeast and filamentous forms. Two identified genes were confirmed to relate to the two phases observed. One of these genes, named UmRrm75, was further characterized by generating a mutant lacking the gene. UmRrm75 mutants were still able to filament in response to acid pH but had altered phenotypes suggesting a role for UmRrm75 in growth rate, morphogenesis and pathogenicity in corn.

Technical Abstract:
Ustilago maydis displays dimorphic growth, alternating between a saprophytic haploid yeast form and a filamentous dikaryon, generated by mating of haploid cells and which is an obligate parasite. Induction of the dimorphic transition of haploid strains in vitro by change in ambient pH has been used to understand the mechanisms governing this differentiation process. In this study we used suppression subtractive hybridization to generate a cDNA library of U. maydis genes up-regulated in the filamentous form induced in vitro at acid pH. Expression analysis using quantitative RT-PCR showed that the induction of two unigenes identified in this library coincided with the establishment of filamentous growth in the acid pH medium. This expression pattern suggested that they were specifically associated to hyphal development rather than merely acid pH-induced genes. One of these genes, UmRrm75, encodes a protein containing three RNA recognition motifs and glycine-rich repeats and was selected for further study. The UmRrm75 gene contains 4 introns, and produces a splicing variant by a 3'-alternative splicing site within the third exon. Mutants deleted for UmRrm75 showed a slower growth rate than wild type strains in liquid and solid media, and their colonies showed a donut-like morphology on solid medium. Interestingly, although delta-UmRrm75 strains were not affected in filamentous growth induced by acid pH and oleic acid, they exhibited reduced mating, post-mating filamentous growth and virulence. Our data suggest that UmRrm75 is probably involved in cell growth, morphogenesis, and pathogenicity in U. maydis.